Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficien
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production Ryosuke Mitsui 1 & Ryosuke Yamada 1 Hiroyasu Ogino 1
&
Takuya Matsumoto 1 & Shizue Yoshihara 2 & Hayato Tokumoto 2 &
Received: 25 March 2020 / Revised: 9 September 2020 / Accepted: 13 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. Key Points • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed. Keywords Saccharomyces cerevisiae . Lactic acid tolerance . Transcriptome analysis . D-Lactic acid production . Non-neutralized fermentation
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10906-3) contains supplementary material, which is available to authorized users. * Ryosuke Yamada [email protected] 1
Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
2
Department of Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
Lactic acid (LA) is a substance with high industrial demand and has been widely used as flavor, acidulant, and preservative in food, cosmetic, and pharmaceutical industries (Djukić-Vuković et al. 2019). The global LA market is expected to reach US$9.8 by 2025 (Singhvi et al. 2018).
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